1. Bischenberg, 23 rd September 2007The GA 2 LEN/EAACI Allergy School Effects of heat treatment and proteolytic enzymes on allergenicity Dr. Montserrat Fernández Rivas

2. M. Fernández Rivas Food allergens In contrast to inhalant allergens (most) food allergens undergo considerable changes in structure and may form complex structures with other food components before they interact with the individual’s immune system. Food processing and digestion modify the allergenicity of foods, in terms of potential to sensitise and/or induce symptoms. Therefore it is of paramount importance to understand the molecular basis of the effects of food processing and digestion (and we have just begun). Furthermore, the role of the food matrix in which an allergen is processed must also be evaluated. Epitopes:  Linear or sequential: binding is not afected by the folding state of the protein  Conformational: binding is disrupted by changes in the protein folding.

3. M. Fernández Rivas Effects of heat treatment Thermolabile proteins: unfolding conformational epitopes  Bet v 1 homologues  Albumins Thermostable proteins:  Limited unfolding and aggregation: 11S, 7S globulins  Resisting unfolding and refolding on cooling: 2S albumins, nsLTP, parvalbumin Covalent modification of proteins: Maillard reactions  Bet v 1 homologues  Peanut allergens, Ara h 1 and 2  nsLTP

4. M. Fernández Rivas Thermolabile proteins: Bet v 1 homologues Birch pollen-fruit-vegetable syndrome Clinical finding: patients react to the fresh fruit but tolerate it cooked. Patients sensitised to intact native Bet v 1 through the inhalant route recognise conformational epitopes on plant foods that are destroyed during cooking (unfolding) Api g 1 and Gly m 4 are more thermostable than their equivalents in Rosaceae fruits: this is in line with reactions induced by cooked celery and soya-based food supplements

5. M. Fernández Rivas Thermolabile proteins: Bet v 1 homologues 1)Fresh sweet cherries from the market 2)cherry fruit juice, 35% fruit content 3)preserved cherries, sweetened 4)cherry jam, 45% fruit content CD spectra of Pru av 1 at different temperatures Scheurer et al. JACI 2004; 114: 900-7

6. M. Fernández Rivas Thermostable proteins: nsLTP CD spectra of Pru av 3 at different temperatures Scheurer et al. JACI 2004; 114: 900-7 1) fresh sweet cherries from the market 2) cherry fruit juice, 35% fruit content 3) preserved cherries, sweetened 4) cherry jam, 45% fruit content Clinical findings: Patients allergic to peach/apple LTP react with the fresh fruit and with processed products (+) OFC with cooked apple Patients allergic to hazelnut-Cor a 8 react after ingestion of roasted hazelnuts Patients allergic to maize LTP react with cooked maize derivatives (polenta, roasted corn, popcorn)

7. M. Fernández Rivas Maillard reactions and nsLTP Effect of heat treatment +/- glucose on Mal d 3 IgE binding potency 20 min 90 o C 2 hr 100 o C Protection of Mal d 3 by the presence of glucose during heating Sancho AI. Allergy 2005;60:1262-8 (+) (-) SPT Mal d 3 heated 60 min 100ºC: no change The Maillard reaction is achemical reaction between an amino acid and a reducing sugar, usually requiring heat.

8. M. Fernández Rivas Maleki SJ. JACI 2000;106:763-8 Maillard reactions and peanut allergenicity Roasting peanuts: increased IgE-binding x90

9. M. Fernández Rivas Maleki SJ. JACI 2000;106:763-8 Maillard reactions and peanut allergenicity Roasting peanuts: increased IgE-binding + stability to digestion Ara h 2 trypsin inhibitor activity is 3.6 fold increased by roasting and protects Ara h 1 against trypsin digestion. Maleki SJ. JACI 2003; 112:190-5

10. M. Fernández Rivas Cherry extract digested with pepsin: (A) Silver stained (B) IgE immunoblot 1)2 hr without pepsin 2)30 sec 3)1 min 4)15 min 5)30 min 6)1 hour 7)2 hours 8)BSA without pepsin 9)BSA 2 hours Effect of proteolytic enzymes on Bet v 1 homologues Scheurer et al. JACI 2004; 114: 900-7

11. M. Fernández Rivas Scheurer S. JACI 2004; 114: 900-7 Pepsin digestion of purified cherry allergens Bet v 1 homologue nsLTP Profilin

12. M. Fernández Rivas Stability of profilin López-Torrejón G. CEA 2005; 35:1065-72. Melon profilin is completely digested in < 1 min Melon profilin is not affected After heating 100ºC 15 min

13. M. Fernández Rivas Stability of profilin Histamine release Untreated rCuc m 2Positive 2/3 SGF digested rCuc m 2Negative 3/3 López-Torrejón G. CEA 2005; 35:1065-72.

14. M. Fernández Rivas In summary There are no clear rules regarding how different allergens respond to heat treatment and proteolytic enzymes, with some as the Bet v 1 homologues having their allergenicity destroyed, whereas for many others it is unaltered. On the other hand, the allergenicity of some foods may even increase following food processing.